Members of the Hedgehog (Hh) family of signaling molecules mediate important developmental processes including patterning, morphogenesis of organs and cell specification. While the Hh pathway has been studied extensively, there is a lack of detailed understanding of the molecular and cellular mechanisms underlying this pathway. Genetic screens in zebrafish have identified several mutants that are defective in Hh signaling. The sonic-you (syu) locus encodes the zebrafish Sonic hedgehog and detour (dtr) and you-too (yot) loci encode the Gli1 and Gli2 zinc finger transcription factors, respectively. Mutations in these loci disrupt forebrain, spinal cord and somite development. A fourth mutant, chameleon (con), displays similar phenotypes as syu, dtr and yot, but the gene product has not been molecularly identified. Phenotypic analysis of con mutants suggests that con has a key function in Hh signaling during spinal cord development. This project has two specific aims: l) I will pursue the cloning of the con gene by using positional cloning, synteny conservation and candidate gene approaches. 2) The site of con action will be determined by genetic mosaic analysis by transplanting wildtype cells into con mutant embryos and vice versa, followed by assaying expression of nk2.2 in the spinal cord. To determine the epistatic relationship of con with members of the Hh signaling pathway, I will ectopically express Hh signaling components in con mutants and analyze marker gene expression in the spinal cord.